1. Field of the Invention
The present invention relates to the field of molecular genetics and its use in the preparation of therapeutic agents effective in vivo in adult tissue. More particularly, stem cells are genetically modified in vivo and then function to induce regeneration and repair of damaged tissue throughout the life span of an animal. More specifically, the present invention relates to the retroviral-mediated transfer of marker genes into adult tissue in which a mitotically-active state of satellite cells has been induced.
Additionally, the present invention relates to methods of treating genetically-transmitted diseases. The invention also relates to methods and therapeutic treatments for regenerating damaged muscle tissue, as a particular method for muscle regeneration in persons with muscular dystrophy is disclosed.
2. Description of Related Art
The insertion of foreign genes or DNA into an organism's genome has become a powerful tool in experimental biology during the past decade. This modification of genetic information has been attempted via the development of a variety of experimental protocols. Many experiments have transferred genes into mammalian cells in culture and into newly fertilized mammalian eggs that then develop into an intact animal. Less commonly, experiments have been performed wherein foreign DNA is inserted into the genome of cells of young or adult animals in situ. The insertion of foreign DNA into the genome of a cell or embryonic tissue has been postulated to be applicable in the treatment of genetic diseases in higher eukaryotes, particularly in humans. Methods currently under extensive study in the manipulation of genes include in vitro DNA transfection techniques, in vivo whole cell injection (i.e., hematopoetic and myoblast cells), and in vivo as well as in vitro retroviral-mediated gene transfer.
The insertion of foreign genes into an organism's genome thus presents a potentially revolutionary method for treating genetic-defect tractable diseases in man. However, many of these genetic diseases remain to be more fully characterized before gene therapy can be used in their treatment.
Some of the more fully characterized examples of genetic-defect diseases identified in the literature include, by way of example, the various forms of muscular dystrophy (see Table 1), diabetes, hemophilia and albinism. The various forms of muscular dystrophic diseases appear below.
TABLE 1 __________________________________________________________________________ The Progressive Muscular Dystrophies Pattern of Muscular Type of Dystrophy Age of Onset Involvement Special Features CK Level Inheritance __________________________________________________________________________ Duchenne Infancy or early Pelvifemoral; Hypertrophy- High X-linked childhood later pectoral pseudohyper- recessive girdle trophy; cardiac involvement; mental retardation Becker Childhood, Pelvifemoral, Cardiac Moderate X-linked adolescence, later pectoral involvement, recessive or early adult girdle sight; mentation normal Emery-Dreifuss Childhood, Humeroperoneal Contractures Moderate X-linked adolescence posterior neck recessive and biceps muscles Landouzy-Dejerine Late Childhood, Facioscapulo- Heart normal, Slight to Dominant adolescence humeral; pelvic mentation moderate girdle-late normal Scapulohumeral Childhood, Spinal and Cardiomyopathy Slight to Dominant of Seitz or adult humeral, later moderate pelvic girdle girdle-late Limb-girdle (Erb) Childhood, Pectoral or pelvic Heart usually Slight to Variable, adolescence, or both normal, moderate recessive, sometimes mentation or dominant adult normal von Graefe- Childhood, Ocular (sparing Kearns-Sayre Slight to Dominant; Fuchs adolescence pupils); later group have moderate Kearne-Sayre facial and other retinitis recessive muscles (slight) pigmentosa, heart block, stunting of growth, and ovarian dysgenesis Oculopharyngeal Middle or late Levators of lids; Slight to Dominant adult other ocular- normal pharyngeal muscles later Myotonic Infancy, Ocular, facial, Cataracts, Slight to Dominant (Steinert) childhood, Stemomastoid, testicular normal adult forearm, atrophy peroneal Congenital Birth, Infancy Pectoral and Mental Slight to Dominant pelvic girdles, retardation, moderate or recessive or diffuse arthrogryposis __________________________________________________________________________
Among the many genetic diseases described, the muscle degenerative, or muscular dystrophy-causing diseases, have experienced significant recent technological advances. For example, the muscular dystrophies have been traced to particular gene defects, the elucidation of which has experienced particularly significant scientific breakthroughs in terms of genetic molecular characterization. The most widely known of the muscular dystrophies are Duchenne's muscular dystrophy and the less severe Becker muscular dystrophy. Both of these genetic diseases are characterized by an inability of the muscle to produce dystrophin, which is a muscle protein. This defect is an x-linked recessive disease potentially caused by a defect in the dystrophin gene. The dystrophin gene has most recently been established as having a close relative gene on human chromosome 6.sup.24.
Prior studies have attempted the use of foreign normal myoblast injection as a form of gene product replacement. For example, foreign myoblasts containing a normal dystrophin gene have been injected into dystrophic tissue to invoke the expression of the dystrophin protein in the muscle tissue. However, this method presents the inherent risk of immune rejection, as well as the necessity of injection at multiple, probably closely-spaced sites.sup.23. Additionally, injection at multiple sites is necessary with such a therapy because dystrophin, like other muscle proteins, tends to remain localized within a single fiber, close to the nuclei from which it was derived. An additional limitation of currently practiced myoblast-injection techniques is the low fusion rate of implanted myoblasts into normal host muscle tissues.
Muscle tissue is characterized by the presence of satellite cells, which are located between the basal lamina and the sarcolemma of the skeletal muscle fibers. Satellite cells in skeletal muscle are dormant stem cells which are present outside the muscle fibers in an adult skeletal muscle. Each of these inactive stem cells can either proliferate into many satellite cells or mature into an embryonic myoblast upon the appropriate stimulation.
By way of example, satellite cells of the muscle have been shown to become stimulated to replicate by muscle damage (Allbrook, 1981; Carlson et al., 1983). It is also possible that strenuous exercise may stimulate cells to become mitotically active and to replicate..sup.27 Chemical damage via bupivacaine injection into mammalian skeletal muscle cells has been shown to result in rapid recovery of the tissue showing maximum proliferative capacity of satellite cells 36-48 hours following damage.sup.6,14. As stem cells, satellite cells function to supply myonuclei to growing fibers in immature animals as well as to provide myogenic cells for muscle regeneration and repair throughout the life of the animal.
Several laboratories have published protocols by which the hematopoietic cells of mice have had new genes introduced with retroviral vectors ex vivo, with subsequent reintroduction of the treated cells into the bone marrow in vivo.sup.22. In this system, marrow cells are obtained from a donor and incubated with a monolayer of vector-generating producer cells. Since marrow cells, unlike the producer cells, do not attach to the culture disk, they are easily recovered after co-cultivation. The hematopoietic cells are then introduced into a recipient animal by intravenous injection. Space in the hematopoietic system to receive the vector-treated marrow must be made usually by lethally irradiating the recipient. While this technique has been widely used experimentally, its use in humans is limited by the recurrent risk of host immune rejection, as current studies on muscle were performed on mdx (immunosuppressed) animals.sup.23. Additionally, low proportion of cells incorporate the normal myoblast dystrophin gene in myoblast transfer procedures. Moreover, multiple injections of myoblasts are typically required to achieve proper dispersion of the myoblasts in situ.
A need thus remains in the art for the development of a therapeutic system which minimizes or eliminates host immune response, perhaps through the development of an entirely in vivo vector-induced, host cell gene incorporation process. However, technical difficulties in the manipulation of genomes from organisms both in achieving the initial incorporation of the desired gene by the host cells as well known as achieving expression of those genes in the host have limited techniques of direct incorporation of a gene without a "carrier" cell into damaged tissue in vivo. DNA transfection and retroviral mediated gene incorporation are two methods of such a "direct gene" incorporation which eliminates the need for a "carrier" cell.
The treatment of genetically-related diseases with techniques as DNA transfection has thus far, unfortunately, not met with great success. In DNA transfection, DNA (presumably which includes a non-defective counterpart of the defective gene) is introduced into cells in culture as part of a coprecipitate with calcium phosphate or dextran sulfate..sup.9 A successful result is a viable cell containing one to many copies of the new gene which continuously expresses the new genetic information.
While several limitations exist in this system, the most significant limitation is that it is a very inefficient means of transferring genes into mammalian cells. For example, only one in a thousand cells (more typically, one cell in a million) will incorporate the newly transformed gene. Additionally, not all cultured cell lines are susceptible to this method of gene transfer. For example, while the stem cells located in bone marrow are typically susceptible to this method of gene transfer, they are present only as a small fraction (less than one cell in a thousand) of the total nucleated cells of the tissue. Another limitation is that it is an ex vivo technique.
This method is therefore inefficient as only a small population of cells incorporate the desired gene, failing to accomplish gene delivery to a large fraction of a target cell population. Even if the technical difficulties of gene incorporation overcome, a second obstacle to the effective employment of this technique in the higher eukaryotes and mammals which has not yet been solved in being able to attain proper expression of successfully incorporated genes by the host.
Retroviral-mediated gene transfer (i.e., the use of retroviruses to deliver genes into cells) is an alternative gene transfer technology which has met with a limited, yet improved, success in host genome incorporation rates. Using this technique, it is now possible to insert a gene into a retroviral vector to obtain a recombinant virus, and then infect target cells with the retrovirus (which includes a particular gene of interest) and achieve the expression of the foreign gene by the host cell's chromosomes. Retroviruses are RNA viruses, that is, the viral genes are encoded in an RNA molecule rather than in a DNA molecule. All RNA tumor viruses are members of the retrovirus family, but not all retroviruses are oncogenic or even pathogenic.
In retroviral-mediated gene transfer, the viral RNA is first converted to DNA when an RNA virus penetrates a cell. If the cell penetrated is a replicating cell (i.e. mitotically active), the DNA will enter the nucleus and integrate into a chromosome. This integrated DNA becomes indistinguishable as far as the cell is concerned from any other cellular gene. It is from this integrated form, that the viral genes are expressed. In this process, integration of the viral genome into the cell's chromosome is an essential part of its replication. However, RNA retroviruses only insert their genome into mitotically active cells; thus making it highly unlikely that an RNA retrovirus would infect a mature myonucleus. Thus, only immature animal tissues have been used experimentally with retroviral-mediated gene transfer systems with much improvement of gene-incorporation rates.
Retroviral mediated gene transfer remains a relatively inefficient gene transfer system for adult tissues owing to low gene incorporation rates of target cells. It would therefore only be by means of a highly efficient retroviral vector system that genetic manipulation of totipotent stem cells would become practical, as currently practiced methods of retroviral-gene transfer provide only a small fraction of genetically-altered cell components.
A number of procedures are known to artificially induce a mitotically-active state in a cell population. For example, exercise,.sup.27 tissue trauma,.sup.6 chemical injection,.sup.28 and radiation.sup.9,30 have been shown to make tissue or cells in culture mitotically active. Many pharmaceutical agents (especially anesthetics, such as Marcaine.RTM.) have been found to destroy or damage muscle fibers..sup.28 For example, it has been found that when bupivacaine (a local anesthetic) is injected into skeletal muscle, existing muscle fibers (including myonuclei) are destroyed,.sup.15,16 curiously, however, the satellite cells in these tissues are left undamaged.
Despite developments in retroviral-mediated gene transfer and studies regarding the in vitro and in vivo induction of mitotically active states in certain cell populations, the many unknown aspects of the structure and function of retroviruses have prevented the combination of these advantages in the development of an efficient gene-transfer system. Technical difficulties still exist in the use of retroviral vectors for gene transfer in large animals (i.e., humans), such as in the efficiency of infection of pluripotent stem cells and in the long-term stability of expressed genes. Moreover, the use of retroviral-mediated gene transfer is limited by the size of the gene constructs which the virus is able to carry. For example, genes having more than 1400 base pairs have been found to be too large for common vectors. Such results in difficulties in replacing proteins, either for functional studies or in gene therapy experiments, which involve genes of this size.
In the development of an efficient retroviral gene transfer system, two parameters must be optimized. First is the capacity to infect a large proportion of the target cells, a property dependent at least in part on the ability to generate a high concentration (or titer) of recombinant virus. Second is the capacity to have the gene expressed properly in the host.
An intense need exists in the art of molecular biology to either circumvent the limitations of retroviral-mediated gene transfer (gene size, gene incorporation ratio in target cells) if retroviral vectors are to be used in the medical treatment of genetic disease in humans. Additionally, the need for an efficient gene transfer system is desirable for its usefulness to basic research, as well as being an absolute prerequisite for application to human therapy..sup.10
The innovative development of gene transfer technology to improve target cell incorporation ratio while achieving successful and acceptable amounts of gene expression in the host, while eliminating host immune-response problems, would revolutionize currently practiced methods of treating genetically-induced maladies, especially those muscle degenerative diseases which have escaped human intervention over the years.